Morphological diversity of indigenous wild pomegranate (Punica granatum L. var. spinosa) accessions from northeast of Iran

Abstract Wild pomegranate is a valuable edible species in the plant ecosystem of the Hyrcanian forests and the northern plains of the Caspian coast in Iran. The genetic diversity of these wild pomegranates can be effective in pomegranate breeding programs and germplasm conservation. In the present study, morphological diversity in 103 wild pomegranates (Punica granatum L. var. spinosa) in the northeastern area of Iran was studied using 46 traits related to trees, flowers, and fruits. The results showed that the fruit weight ranged from 17.93 to 99.9 g with an average of 48.92 g, the total aril weight ranged from 0.54 g to 64.78 g with an average of 24.25 g, and the weight of 100 arils was between 4.89 and 46.21 with an average of 13.79. The fruit cracking percent, crown shape, aril juiciness, calyx, and corolla colors show a high coefficient of variation (CV > 70%). Based on PCA results, fruit weight and total aril weight, peel weight, and fruit length and diameter were important on determining differences among accessions. In biplot analysis, genotype distribution was determined by two main factors. In cluster analysis, the studied accessions were divided into two different major clusters and two subclusters in each one. The results showed a high diversity of important pomological traits in wild pomegranates such as fruit weight, fruit cracking percent, crown shape, total aril weight, aril juiciness corolla, and calyx color that can be used in breeding programs to improve pomegranate juice quality and marketability.

The harvested fruits from these trees are used by local people to prepare various pomegranate pastes. These pastes are an essential part of the local cuisine, such as vegetable processing, sour chicken, Fosanjan, and conserved olives, and they are also considered tasting kebab (Mirjalili & Poorazizi, 2015). In addition, these fruits are noted as a great source of natural antioxidants and health-promoting constituents like anthocyanins, phenolics, and flavonoids (Thakur et al., 2020) and they have some antitumoral effects (Sineh Sepehr et al., 2012).
The most critical step in any breeding program is screening and identifying superior genotypes, which is very costly and timeconsuming. Diversity is the basis of all selection and crop improvement requires genetic diversity. As the diversity increases, the selection scope also extends. It is better to identify and screen superior genotypes in the same climatic region as a collection. However, wild genotypes do not have acceptable yields, so they have intrigue breeders. Still, they are valuable in some quantitative and quality traits such as resistance to abiotic and biotic stresses and nutritional quality (Zuriaga et al., 2022). Wild pomegranates have broad geographical distributions from Iran and Turkmenistan to northern India (Chandra et al., 2010). In Iran, wild pomegranates are adapted very well to various environmental conditions, and they are dispersed around all of the plains in coast Caspian and Hyrcanian forests (Khadivi et al., 2020). Today, with human exploitation in Iran, the natural resources of wild pomegranate populations are often endangered (Khan et al., 2014). Hence, studies on the wild pomegranate diversity are valuable to find superior genotypes for breeding programs and to establishing core collections for germplasm conservations and more detailed studies (Arlotta et al., 2022).
Numerous studies have been conducted on the morphological diversity of fruits and the biochemical composition of domestic and wild pomegranates in different parts of the world. For example, in a study on morphological variability of wild pomegranate in northern parts of Iran, Khadivi et al. (2020) reported a high diversity in fruit weight, fruit peel color, aril color, fresh arils weight, and total soluble solids. A wide variation in fruit size, fruit peel, anthocyanin content, total soluble solids, aril juice content, and seed hardness was reported in cultivated pomegranates in Iran (Sarkhosh et al., 2009). Similar phenotypic variation has been reported within wild pomegranate collections in Pakistan (Aziz et al., 2020;Nafees et al., 2015) and India (Mishra et al., 2016;Singh & Gupta, 2018). A developing of SSRs was introduced as a rapid tool for the identification of pomegranate cultivars in the study of nutraceutical and genetic diversity of novel pomegranate genotypes in comparison to leading commercial pomegranate varieties (Arlotta et al., 2022). In addition, the genetic variability in pomegranate cultivars was reported using both nuclear (SSRs) and chloroplast genetic regions (trnH-psbA spacer, and matk gene; Shahsavari et al., 2022). However, diversity in flower characteristics has received less attention.
The morphological diversity of wild pomegranates from the northeast region of Iran is lacking. The objective of the current study was to determine the morphological variability of trees, flowers, and fruits among wild pomegranate accessions in the northeast region of Iran to inform best strategies for incorporating beneficial traits into pomegranate breeding programs as well as aid germplasm conservation efforts.

| Plant materials
To study the phenotypic variability of wild pomegranate in the northeastern area of Iran, 103 wild pomegranate accessions (P. granatum var spinosa) from natural habitats of Semnan, Mazandaran, and Golestan provinces were assessed using pomological and morphological traits. Geographical coordinates and altitude corresponding to each surveyed area are presented in Figure 1 and Table 1. To avoid the possibility of sampling and collecting clones of the selected accessions, at least a 150 m distance was considered between the accessions in each collection site.

| Measured traits
Forty-six morphological characteristics related to trees, leaves, thorns, flowers, and fruits were evaluated based on the pomegranate descriptor (UPOV, 2012). At first, the geographical coordinates and altitude of each wild pomegranate accession were recorded with a GPS device, and coding was done for each tree (Figure 1). Tree height was measured with an index (Figure 2a), and the characteristics related to tree vigor, growing habits, type of flowers, and thorns were evaluated based on the respective descriptor. In total, 25 fullmaturity fruits, 25 leaves, and 25 flowers were randomly sampled to assess each accession. The traits, including fruit weight, aril weight, and peel weight, were measured using an electronic laboratory balance with 0.01 g precision (Bonvoisin, US), and fruit crown length and diameter, fruit crown width, aril length and diameter, peel thickness, leaf length and width, petiole length, and fruit length and diameter were measured using a digital caliper (Insize Co.). Flower-related traits were recorded in the first series of bell-shaped flowers (fertile flowers) in late spring. Fruit-related traits were assessed in fully ripened fruit in autumn.

| Statistical analysis
Descriptive analysis of each trait, including minimum, maximum, mean, variance, and coefficient of variation (CV%: SD/mean*100), was analyzed by SPSS software version 22 (SPSS Inc., Chicago, II., US). Relationships among the accessions were investigated using principal component analysis (PCA) using PAST software. Cluster analysis was also done by PAST software with the Ward method.
The calculation of distances was performed after Z-score standardization. A scatter plot among populations was created according to the first two principal components (PC1 and PC2) using PAST software. The heatmap clustering was drawn by "heatmap" packages in Rstadio ver.1.0.136 software.

| Morphological traits description
A description of the analysis of morphological traits of 103 wild pomegranate accessions was presented in Table 2. The results showed significant differences among the studied accessions based on all the characters recorded (p < .01). The high coefficient of variance (CVs) of a trait indicates excessive diversity in the population, indicating its higher opportunity in selection at a breeding program.
The range of CVs was from 9.04 (in calyx length-to-width ratio) to 292% (in cracking percent), with an average of 35.97% ( Table 2).
Among the studied traits, in order, fruit cracking percent, crown shape, corolla color, calyx color, aril juiciness, aril color, length of thorn, weight of arils in a fruit, weight of 100 arils, flower shape, carob moth, aril hardiness, fruit over color, and fruit weight have the high CVs (more than 39%).
The fruit weight with means of 48.92 g ranged from 17.93 g (SA8) to 99.90 g (SH11). The fruit weight in Iranian cultivated pomegranates is 103.28-407.59 g (Khadivi & Arab, 2021), which indicates that wild pomegranate accessions predominantly are smaller in size than commercial varieties ( Figure 2). The fruit weight in wild pomegranates from India has been reported in the range 80.50-85.17 g (Kher, 1999), and 55.10-83.50 g with averages of 64 g (Thakur et al., 2011). Khadivi et al. (2020) reported that the fruit weight of wild pomegranates in Iran ranged from 19.20 to 185.00 g with an average of 59.89 g. Genetic variation and pedoclimatic conditions can influence fruit weight (Martinez et al., 2006). In this study, since all the genotypes were evaluated in the different geographical zone, variation in fruit weight can be influenced by genetic and climatic conditions.
The weight of arils in a fruit ranges from 0.54 (AZAD2) to 64.87 g (SH11) with a mean of 24.25 g. The weight of 100 arils ranged from 4.89 to 46.21 g (MIN3), with a mean of 13.79 g. It is a standard for determining the size of the aril so that the heavier the weight of 100 arils, the larger the arils, and vice versa. The total weight of pomegranate aril is considered one of the most important economic criteria for the industrial production of pomegranates (Maestre et al., 2000).

Biplot analysis represented a two-dimensional distribution
The AZAD2 with the lowest aril weight on the first plot side, MIN11 with high fruit weight (98.24 g) on the second plot side, and SH7, SH10, and SH11 with large fruits and highest total aril weight on the third plot side were the further away from other accessions.
The studied wild pomegranate accessions were separable in four biplot regions ( Figure 5). In region 1, there are accessions from Amol and MIN (1). The fruits of these accessions were longer and they had larger arils than other wild pomegranates. In the fourth biplot area, there are accessions from Gor (3, 5, 7, 9, and 11-14), Azad (1, 3, 4, 10, 11, 14, and 15), SH (2-6), SA (4, 11, and 13), MIN (4, 6, and 8), and Gol (2, 6, and 9). The predominant accessions in this group were from the Gorgan region, and they are similar in fruit crown length and width and petal length and width. The information related to samples and variables of a data matrix is displayed geographically in a biplot.
Variables are indicated as vectors, linear axes, or nonlinear trajectories, while samples are shown as points. These points may be used to present the level of a categorical variable (Khadivi et al., 2020).

| Cluster analysis
Cluster analysis is a technique to group a set of observations in such a way that individuals in the same cluster are more similar to each total weight of arils, peel weight, fruit diameter, aril percentage, the weight of 100 arils, and fruit length. The cluster analysis groups are largely confirmed with PCA results.

| Scatter plot of populations
Scatter plot analysis was performed to show the distribution of populations based on two main principal components (Figure 7).
The vicinity of populations in the plot indicates their genetic similarity, and the high distance indicates high morphological diversity.
As seen in the scatter plot, the Amol population was located next to the Miankaleh population in zone 1. Sari, Gorgan, and Azadshahr were located next to each other in the fourth scatter plot area. The Golestan population was in zone 2 of the scatter plot with a short distance from the Sari population. Shahroud population was located in zone 3, far from other populations. Shahroud region, with high altitude, is geographically separated from other studied areas (Figure 1).

| Heatmap analysis
The heatmap is a technique that visualizes the magnitude of a phenomenon in color in two dimensions. This analysis shows how aril color, aril juice, aril diameter, and aril length and weight of 100 arils, whereas the second subgroup (C*II) consists of corolla color, calyx color, tip leaf shape, fruit length/width ratio, petiole length, the thickness of peel, croon diameter, crown shape, fruit length of the crown, carob moth, aril percent, aril length/width ratio, aril hardiness, fruit width of the crown, petal width, petal length, leaf number per node, leaf width, calyx length/width ratio, and fruit over color ( Figure 8). A total of 50% of the traits were classified as the third group and were found in the second subgroup (C*II), so these traits showed relatively high CVs, and thus, possessed high genetic variation. One of the effective factors for achieving maximum heterosis in breeding programs would be considering two completely inconsistent parents, one with the highest and the other with the lowest trait value (Ebrahimi & Alipour, 2020;El-Sayed & Abbas, 2015).
Grouping and characterizing germplasm provide valuable information for breeders and eliminates population resampling steps. One of the most common methods used by breeders in their breeding programs is selection along with generation testing. Access to heterosis and genetic recombination among the population is one of the success factors in the selection program. The possibility of heterosis in cross-breeding programs with a rising genetic distance has been reported in many studies. Indeed, crosses occurring between genotypes at greater genetic distances can certainly lead to more recombination and heterosis (Subramanian & Subbaraman, 2010;Usharani et al., 2015). Grouping genotypes based on genetic distance can be a practical approach to breeding multiple traits.

| CON CLUS ION
In this study, the genetic variation of wild pomegranate in the north and northeast of Iran was evaluated by morphological traits. The results showed that the examined area has a relatively large genetic diversity. There are high variations in fruit cracking percent, crown shape, corolla color, calyx color, aril juiciness, aril color, length of thorn, the weight of arils in a fruit, weight of 100 arils, flower shape, susceptibility to carob moth, aril hardiness, fruit over color, and fruit weight in wild pomegranates. Our finding revealed superior accessions, including SH11, AZAD2, MIN3, MIN11, SA11, and GOL3, which can be applied to these as parents that certainly increase the chance of obtaining desirable genotypes in a breeding program. These accessions are of great value for most of the quantitative traits. The results provide valuable information about the identification of superior genotypes, genetic diversity, and the distribution of wild pomegranate genotypes. The results of this study provide information that can be effective in breeding programs for the development of superior genotypes as well as germplasm conservation programs and preventing the genetic drift in indigenous wild pomegranates in natural habitats.

ACK N OWLED G M ENT
The authors are responsible for the content and writing of the study.

CO N FLI C T O F I NTE R E S T
The authors have no conflict of interest.

DATA AVA I L A B I L I T Y S TAT E M E N T
The data that support the findings of this study are available from the corresponding author upon reasonable request.

E TH I C A L A PPROVA L
This study does not involve any human or animal testing.